New ras-like protein

ABSTRACT

The present invention provides a human Ras-like protein (RLP) and polynucleotides which identify and encode RLP. The invention also provides expression vectors, host cells, agonists, antibodies and antagonists. In addition, the invention provides methods for producing RLP and for treating or preventing disorders associated with expression of RLP.

[0001] This application is a continuation of co-pending U.S. applicationSer. No. 09/198,559, filed on Nov. 23, 1998, entitled New Ras-likeProtein, the disclosure of which is incorporated by reference.

[0002] U.S. application Ser. No. 09/198,559 is a divisional of U.S.application Ser. No. 08/846,790, filed on Apr. 30, 1997, now U.S. Pat.No. 5,973,130, issued on Oct. 26, 1999, the disclosure of which isincorporated by reference.

FIELD OF THE INVENTION

[0003] This invention relates to nucleic acid and amno acid sequences ofa new human Ras-like protein and to the use of these sequences in thediagnosis, prevention, and treatment of disorders associated with cellproliferation and inflammation.

BACKGROUND OF THE INVENTION

[0004] Guanine nucleotide-binding proteins (GTP-binding proteins, or Gproteins) participate in a wide range of regulatory functions includingmetabolism, growth, differentiation, signal transduction, cytoskeletalorganization, and intracellular vesicle transport and secretion. Theseproteins control a diverse sets of regulatory pathways in response tohormones, growth factors, neuromodulators, or other signaling molecules.When these molecules bind to transmembrane receptors, signals arepropagated to effector molecules by intracellular signal transducingproteins. Many of these signal transducing proteins are members ofGTP-binding proteins.

[0005] Low molecular weight (LMW) GTP-binding proteins are smallproteins which consist of single polypeptides of 21-30 kDa. Theseproteins regulate cell growth, cell cycle control, protein secretion,and intracellular vesicle interaction. In particular, the LMWGTP-binding proteins activate cellular proteins by transducing mitogenicsignals involved in various cell functions in response to extracellularsignals from receptors (Tavitian, A. (1995) C. R. Seances Soc. Biol.Fil. 189:7-12). During this process, the hydrolysis of GTP acts as anenergy source as well as an on-off switch for the GTPase activity of theLMW GTP-binding proteins.

[0006] The LMW GTP-binding proteins are classified into fivesubfamilies: Ras, Rho, Ran, Rab, and ADP-ribosylation factor. Despitetheir sequence variations, all five subfamilies share common conservedstructural features. Four sequence regions, termed motifs I-IV, areconserved in the LMW GTP-binding proteins. Motif I is the most variableand has the signature, GXXXXGK. The lysine residue is essential ininteracting with the β- and γ-phosphates of GTP. Motif II, III, and IVare highly conserved, with DTAGQE, NKXD, and EXSAX as their respectivesignatures. These motifs regulate the binding of γ-phosphate, GTP, andthe guanine base of GTP, respectively. Most of the membrane bound LMWGTP-binding proteins generally require a carboxy terminal isoprenylgroup for membrane association and biological activity. The isoprenylgroup is added posttranslationally by a mechanism which recognizes aterminal cysteine residue alone or a terminal cysteine-aliphatic aminoacid-aliphatic amino acid-any amino acid (CAAX) motif. Additionalmembrane-binding energy is often provided by either internalpalmitoylation or a carboxy terminal cluster of basic amino acids. TheLMW GTP-binding proteins also have a variable effector region, locatedbetween motifs I and II, which is characterized as the interaction sitefor guanine nucleotide exchange factors (GEFs) or GTPase activatingproteins (GAPs). GEFs induce the release of GDP from the active form ofthe G protein, whereas GAPs interact with the inactive form bystimulating the GTPase activity of the G protein.

[0007] The Ras subfaily proteins already indicated supra are essentialin transducing signals from receptor tyrosine kinases (RTKs) to a seriesof serine/threonine kinases which control cell growth anddifferentiation. Mutant Ras proteins, which bind but cannot hydrolyzeGTP, are permanently activated, and cause continuous cell proliferationor cancer. TC21, a Ras-like protein, is found to be highly expressed ina human teratocarcinoma cell line (Drivas, G. T. et al. (1990) Mol.Cell. Biol. 10: 1793-1798). Rin and Rit are characterized asmembrane-binding, Ras-like proteins without the lipid-binding CAAX motifand carboxy terminal cysteine (Lee, C.-H. J. et al. (1996) J. Neurosci.16: 6784-6794). Further, Rin is shown to localize in neurons and havecalcium-dependant calmodulin-binding activity.

[0008] The discovery of a new human Ras-like protein and thepolynucleotides which encode it satisfies a need in the art by providingnew compositions which are useful in the diagnosis, prevention, andtreatment of disorders associated with cell proliferation andinflammation.

SUMMARY OF THE INVENTION

[0009] The present invention features a human Ras-like proteinhereinafter designated RLP and characterized as having similarity to ahuman and a mouse Ras-like protein, TC21 and Rit, respectively.

[0010] Accordingly, the invention features a substantially purified RLPhaving the amino acid sequence shown in SEQ ID NO: 1.

[0011] One aspect of the invention features isolated and substantiallypurified polynucleotides that encode RLP. In a particular aspect, thepolynucleotide is the nucleotide sequence of SEQ ID NO:2.

[0012] The invention also relates to a polynucleotide sequencecomprising the complement of SEQ ID NO:2 or variants thereof. Inaddition, the invention features polynucleotide sequences whichhybridize under stringent conditions to SEQ ID NO:2.

[0013] The invention additionally features nucleic acid sequencesencoding fragments, or the complement of the polynucleotide sequences,as well as expression vectors and host cells comprising polynucleotidesthat encode RLP. The present invention also features antibodies whichbind specifically to RLP, and pharmaceutical compositions comprisingsubstantially purified RLP. The invention also features methods forstimulating cell proliferation using RLP, and for treating or preventinga disorder associated with cell proliferation or inflammation using anantagonist of RLP.

BRIEF DESCRIPTION OF THE FIGS.

[0014] Figures 1A, 1B, and 1C show the amino acid sequence (SEQ IDNO: 1) and nucleic acid sequence (SEQ ID NO:2) of RLP. The alignment wasproduced using MACDNASIS PRO software (Hitachi Software Engineering Co.,Ltd., San Bruno, Calif.).

[0015]FIG. 2 shows the amino acid sequence alignments among RLP (SEQ IDNO: 1), a human Ras-like protein, TC21 (GI 190877; SEQ ID NO:3), and amouse Ras-like protein, Rit (GI 1656005; SEQ ID NO:4). The alignment wasproduced using the multisequence alignment program of DNASTAR software(DNASTAR Inc, Madison Wis.).

[0016]FIGS. 3A and 3B show the hydrophobicity plots (MACDNASIS PROsoftware) for RLP (SEQ ID NO: 1) and the human Ras protein (SEQ IDNO:3), respectively. The positive X axis reflects amino acid position,and the negative Y axis, hydrophobicity.

DESCRIPTION OF THE INVENTION

[0017] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0018] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

[0019] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

[0020] Definitions

[0021] “Nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide, or polynucleotide, and fragments orportions thereof, and to DNA or RNA of genomic or synthetic origin whichmay be single- or double-stranded, and represent the sense or antisensestrand. Similarly, “amino acid sequence” as used herein refers to anoligopeptide, peptide, polypeptide, or protein sequence, and fragmentsor portions thereof, and to naturally occurring or synthetic molecules.

[0022] Where “amino acid sequence” is recited herein to refer to anamino acid sequence of a naturally occurring protein molecule, “aminoacid sequence” and like terms, such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

[0023] “Peptide nucleic acid”, as used herein, refers to a moleculewhich comprises an oligomer to which an amino acid residue, such aslysine, and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen, P. E. et al. (1993)Anticancer Drug Des. 8:53-63).

[0024] “RLP”, as used herein, refers to the amino acid sequences ofsubstantially purified RLP obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

[0025] “Consensus”, as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, or which has beenextended using XL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5′ and/orthe 3′ direction and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte clone using the GELVIEW™Fragment Assembly system (GCG, Madison, Wis.), or which has been bothextended and assembled.

[0026] A “variant” of RLP, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

[0027] A “deletion”, as used herein, refers to a change in either aminoacid or nucleotide sequence in which one or more amino acid ornucleotide residues, respectively, are absent.

[0028] An “insertion” or “addition”, as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid or nucleotide residues, respectively, as compared tothe naturally occurring molecule.

[0029] A “substitution”, as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0030] The term “biologically active”, as used herein, refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic RLP, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0031] The term “agonist”, as used herein, refers to a molecule which,when bound to RLP, causes a change in RLP which modulates the activityof RLP. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to RLP.

[0032] The terms “antagonist” or “inhibitor”, as used herein, refer to amolecule which, when bound to RLP, blocks or modulates the biological orimmunological activity of RLP. Antagonists and inhibitors may includeproteins, nucleic acids, carbohydrates, or any other molecules whichbind to RLP.

[0033] The term “modulate”, as used herein, refers to a change or analteration in the biological activity of RLP. Modulation may be anincrease or a decrease in protein activity, a change in bindingcharacteristics, or any other change in the biological, functional orimmunological properties of RLP.

[0034] The term “mimetic”, as used herein, refers to a molecule, thestructure of which is developed from knowledge of the structure of RLPor portions thereof and, as such, is able to effect some or all of theactions of the Ras-like protein.

[0035] The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding RLP or the encoded RLP.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

[0036] The term “substantially purified”, as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

[0037] “Amplification” as used herein refers to the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0038] The term “hybridization”, as used herein, refers to any processby which a strand of nucleic acid bonds with a complementary strandthrough base pairing.

[0039] The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

[0040] The terms “complementary” or “complementarity”, as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A”. Complementaritybetween two single-stranded molecules may be “partial”, in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands.

[0041] The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

[0042] As known in the art, numerous equivalent conditions may beemployed to comprise either low or high stringency conditions. Factorssuch as the length and nature (DNA, RNA, base composition) of thesequence, nature of the target (DNA, RNA, base composition, presence insolution or immobilization, etc.), and the concentration of the saltsand other components (e.g., the presence or absence of formanide,dextran sulfate and/or polyethylene glycol) are considered and thehybridization solution may be varied to generate conditions of eitherlow or high stringency different from, but equivalent to, the abovelisted conditions.

[0043] The term “stringent conditions”, as used herein, is the“stringency” which occurs within a range from about Tm-5° C. (5° C.below the melting temperature (Tm) of the probe) to about 20° C. to 25°C. below Tm. As will be understood by those of skill in the art, thestringency of hybridization may be altered in order to identify ordetect identical or related polynucleotide sequences.

[0044] The term “antisense”, as used herein, refers to nucleotidesequences which are complementary to a specific DNA or RNA sequence. Theterm “antisense strand” is used in reference to a nucleic acid strandthat is complementary to the “sense” strand. Antisense molecules may beproduced by any method, including synthesis by ligating the gene(s) ofinterest in a reverse orientation to a viral promoter which permits thesynthesis of a complementary strand. Once introduced into a cell, thistranscribed strand combines with natural sequences produced by the cellto form duplexes. These duplexes then block either the furthertranscription or translation. In this manner, mutant phenotypes may begenerated. The designation “negative” is sometimes used in reference tothe antisense strand, and “positive” is sometimes used in reference tothe sense strand.

[0045] The term “portion”, as used herein, with regard to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from four amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ IDNO:1” encompasses the full-length human RLP and fragments thereof.

[0046] “Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

[0047] The term “antigenic determinant”, as used herein, refers to thatportion of a molecule that makes contact with a particular antibody(i.e., an epitope). When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0048] The terms “specific binding” or “specifically binding”, as usedherein, in reference to the interaction of an antibody and a protein orpeptide, mean that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words, the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A”, the presence of aprotein containing epitope A (or free, unlabeled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

[0049] The term “sample”, as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acid encoding RLP orfragments thereof may comprise a cell, chromosomes isolated from a cell(e.g., a spread of metaphase chromosomes), genomic DNA (in solution orbound to a solid support such as for Southern analysis), RNA (insolution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

[0050] The term “correlates with expression of a polynucleotide”, asused herein, indicates that the detection of the presence of ribonucleicacid that is similar to SEQ ID NO:2 by northern analysis is indicativeof the presence of MRNA encoding RLP in a sample and thereby correlateswith expression of the transcript from the polynucleotide encoding theprotein.

[0051] “Alterations” in the polynucleotide of SEQ ID NO: 2, as usedherein, comprise any alteration in the sequence of polynucleotidesencoding RLP including deletions, insertions, and point mutations thatmay be detected using hybridization assays. Included within thisdefinition is the detection of alterations to the genonic DNA sequencewhich encodes RLP (e.g., by alterations in the pattern of restrictionfragment length polymorphisms capable of hybridizing to SEQ ID NO:2),the inability of a selected fragment of SEQ ID NO: 2 to hybridize to asample of genomic DNA (e.g., using allele-specific oligonucleotideprobes), and improper or unexpected hybridization, such as hybridizationto a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding RLP (e.g., using fluorescent in situhybridization [FISH] to metaphase chromosomes spreads).

[0052] As used herein, the term “antibody” refers to intact molecules aswell as fragments thereof, such as Fa, F(ab′)₂, and Fv, which arecapable of binding the epitopic determinant. Antibodies that bind RLPpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or peptide used to immunize an animal can be derived fromthe transition of RNA or synthesized chemically, and can be conjugatedto a carrier protein, if desired. Commonly used carriers that arechemically coupled to peptides include bovine serum albumin andthyroglobulin. The coupled peptide is then used to immunize the animal(e.g., a mouse, a rat, or a rabbit).

[0053] The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

[0054] The Invention

[0055] The invention is based on the discovery of a new human Ras-likeprotein (RLP), the polynucleotides encoding RLP, and the use of thesecompositions for the diagnosis, prevention, or treatment of disordersassociated with cell proliferation and inflammation.

[0056] Nucleic acids encoding the human RLP of the present inventionwere first identified in Incyte Clone 683101 from an uterine tissue cDNAlibrary (UTRSNOT02) through a computer search for amino acid sequencealignments. A consensus sequence, SEQ ID NO:2, was derived from thefollowing overlapping and/or extended nucleic acid sequences: IncyteClones 1282862 (COLNNOT16), 1501943 (SINTBST01), and 683101 (UTRSNOT02).

[0057] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO: 1, as shown in FIGS.1A, 1B and 1C. RLP is 199 amino acids in length It has four conservedGTP-binding sites encompassing residues G13-K19, D60-E65, N118-D121, andE146-C150, which are analogous to those of other LMW GTP-bindingproteins. The effector binding region of RLP encompasses residuesD16-R24. Unlike most known members of the Ras proteins, the carboxyterminus of RLP lacks either a CAAX motif or a cysteine residue forlipid-binding. However, RLP has a carboxy terminal cluster of basicresidues and a potential EF-hand calcium-binding domain encompassingresidues D121-G133. These features are similar to the Ras-like proteins,Rit (GI 1656005) and Rin (Lee et al., supra), suggesting that RLP is amembrane-associated protein with a potential calcium-dependent function.Additionally, RLP has two potential N-glycosylation sites at N110 andN155, two cAMP- and cGMP-dependent protein kinase phosphorylation sitesat R178-S181 and R179-S182, four potential casein kinase IIphosphorylation sites at T48-D51, S91E94, S129-E132, and T189-E192, andfour protein kinase C phosphorylation sites at T28-R30, T42-R44,T87-R89, and T177-R179. As shown in FIG. 2, RLP has chemical andstructural homology with a hurnan Ras-like protein, TC21 (GI 190877; SEQID NO:3) and a mouse Ras-like protein, Rit (GI 1656005; SEQ ID NO 4). Inparticular, RLP shares 47% identity with the Ras-like proteins, TC21 andRit. As illustrated by FIGS. 3A and 3B, RLP and TC21 have rather similarhydrophobicity plots with the exception of their carboxy terminus.Northern analysis shows the expression of RLP in various cDNA libraries,at least 34% of which are immortalized or cancerous and at least 67% ofwhich either involve immune response or are expressed in fetal/infanttissues or organs.

[0058] The invention also encompasses RLP variants. A preferred RLPvariant is one having at least 80%, and more preferably 90%, amino acidsequence identity to the RLP amino acid sequence (SEQ ID NO: 1). A mostpreferred RLP variant is one having at least 95% amino acid sequenceidentity to SEQ ID NO: 1.

[0059] The invention also encompasses polynucleotides which encode RLP.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of RLP can be used to generate recombinant molecules whichexpress RLP. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 asshown in FIGS. 1A, 1B and 1C.

[0060] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding RLP, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring RLP, and all such variations are to beconsidered as being specifically disclosed.

[0061] Although nucleotide sequences which encode RLP and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring RLP under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding RLP or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding RLP and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0062] The invention also encompasses production of DNA sequences, orportions thereof, which encode RLP and its derivatives, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art at the time of thefiling of this application. Moreover, synthetic chemistry may be used tointroduce mutations into a sequence encoding RLP or any portion thereof.

[0063] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed nucleotide sequences, andin particular, those shown in SEQ ID NO:2, under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding conplex or probe, as taughtin Wal, G. M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) andKimmel, A. R. (1987; Methods Enzymol. 152:507-511), and may be used at adefined stringency.

[0064] Altered nucleic acid sequences encoding RLP which are encompassedby the invention include deletions, insertions, or substitutions ofdifferent nucleotides resulting in a polynucleotide that encodes thesame or a functionally equivalent RLP. The encoded protein may alsocontain deletions, insertions, or substitutions of amino acid residueswhich produce a silent change and result in a functionally equivalentRLP. Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of RLP is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid;positively charged amino acids may include lysine and arginine; andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine;phenylalanine and tyro sine.

[0065] Also included within the scope of the present invention arealleles of the genes encoding RLP. As used herein, an “allele” or“allelic sequence” is an alternative form of the gene which may resultfrom at least one mutation in the nucleic acid sequence. Alleles mayresult in altered mRNAs or polypeptides whose structure or function mayor may not be altered. Any given gene may have none, one, or manyallelic forms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0066] Methods for DNA sequencing which are well known and generallyavailable in the art may be used to practice any embodiments of theinvention. The methods may enploy such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE (US Biochemical Corp, Cleveland, OH), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of recombinant polymerases andproofreading exonucleases such as the ELONGASE Amplification Systemmarketed by Gibco BRL (Gaithersburg, Md.). Preferably, the process isautomated with machines such as the Ha m ilton Micro Lab 2200(Harmilton, Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research,Watertown, Mass.) and the ABI 377 DNA sequencers (Perkin Elmer).

[0067] The nucleic acid sequences encoding RLP may be extended utilizinga partial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to linker sequence and a primer specific to the knownregion. The amplified sequences are then subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

[0068] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO4.06 Primer Analysis software (National Biosciences Inc., Plymouth,Minn.), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

[0069] Another method which may be used is capture PCR which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR.

[0070] Another method which may be used to retrieve unknown sequences isthat of Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

[0071] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA.

[0072] Geno mnic libraries may be useful for extension of sequence intothe 5′ and 3′ non-transcribed regulatory regions.

[0073] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled deviCe camera. Output/light intensity may be converted toelectrical signal using appropriate software (e.g. GENOTYPER andSEQUENCE NAVIGATOR, Perkin Elmer) and the entire process from loading ofsamples to computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable for thesequencing of small pieces of DNA which might be present in limitedamounts in a particular sample.

[0074] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode RLP, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of RLP in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and expressRLP. As will be understood by those of skill in the art, it may beadvantageous to produce RLP-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

[0075] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterRLP encoding sequences for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, or introduce mutations, and so forth

[0076] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding RLP may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of RLP activity, it may be useful toencode a chimeric RLP protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the RLP encoding sequence and theheterologous protein sequence, so that RLP may be cleaved and purifiedaway from the heterologous moiety.

[0077] In another embodiment, sequences encoding RLP may be synthesized,in whole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res.

[0078] Symp. Ser. 215-223, Hom, T. et al. (1980) Nucl. Acids Res. Symp.Ser. 225-232). Alternatively, the protein itself may be produced usingchemical methods to synthesize the amino acid sequence of RLP, or aportion thereof. For example, peptide synthesis can be performed usingvarious solid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431A Peptide Synthesizer (Perkin Elmer).

[0079] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W H Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of RLP, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0080] In order to express a biologically active RLP, the nucleotidesequences encoding RLP or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0081] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding RLPand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

[0082] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding RLP. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0083] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector--enhancers, promoters, 5′ and 3′untranslated regions--which interact with host cellular proteins tocarry out transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene,LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In ma mimalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding RLP,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

[0084] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for RLP. For example, whenlarge quantities of RLP are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asthe BLUESCRIPT phagemid (Stratagene), in which the sequence encoding RLPmay be ligated into the vector in frame with sequences for the a mino-terminal Met and the subsequent 7 residues of B-galactosidase so that ahybrid protein is produced; pIN vectors (Van Heeke, G. and S. M.Schuster (1989) J. Biol. Cheim 264:5503-5509); and the like. pGEXvectors (Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0085] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0086] In cases where plant expression vectors are used, the expressionof sequences encoding RLP may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R.et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) ResultsProbl. Cell Differ. 17:85-105). These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. Such techniques are described in a nurnber of generallyavailable reviews (see, for example, Hobbs, S. or Murry, L. E. in McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New York,N.Y.; pp. 191-196).

[0087] An insect system may also be used to express RLP. For example, inone such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding RLPmay be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of RLP will render the polyhedrin gene inactive andproduce recombinant virus lacking coat protein. The recombinant virusesmay then be used to infect, for example, S. frugiperda cells orTrichoplusia larvae in which RLP may be expressed (Engelhard, E. K etal. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

[0088] In mamalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding RLP may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing RLP in infected host cells (Logan, J. andShenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mamalian host cells.

[0089] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding RLP. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding RLP, its initiation codon, and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a portion thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et a]. (1994) Results Probl. Cell Differ.20:125-162).

[0090] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not li mited to, acetylation,carboxylation, glycosylation, phosphorylation, lipidation, andacylation. Post-translational processing which cleaves a “prepro” formof the protein may also be used to facilitate correct insertion, foldingand/or function. Different host cells such as CHO, HeLa, MDCK, HEK293,and W138, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

[0091] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress RLP may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

[0092] Any number of selection systems m may be used to recovertransformed cell lines. These include, but are not limited to, theherpes simplex virus thymidine kinase (Wigler, M. et al. (1977) Cell11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al. (1980)Cell 22:817-23) genes which can be employed in tk⁻or aprt⁻cells,respectively. Also, antimetabolite, antibiotic or herbicide resistancecan be used as the basis for selection; for example, dhfr which confersresistance to methotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad -Sci. 77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to c blorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpβ,which allows cells to utilize in dole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. 20 Biol. 55:121-131).

[0093] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding RLP isinserted within a marker gene sequence, recombinant cells containingsequences encoding RLP can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding RLP under the control of a single promoter. Expressionof the marker gene in response to induction or selection usuallyindicates expression of the tandem gene as well.

[0094] Alternatively, host cells which contain the nucleic acid sequenceencoding RLP and express RLP may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not l i mited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

[0095] The presence of polynucleotide sequences encoding RLP can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or portions or fragments of polynucleotides encoding RLP. Nucleicacid amplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding RLP to detect transformantscontaining DNA or RNA encoding RLP. As used herein “oligonucleotides” or“oligomers” refer to a nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides, preferably about 15 to30 nucleotides, and more preferably about 20-25 nucleotides, which canbe used as a probe or amplimer.

[0096] A variety of protocols for detecting and measuring the expressionof RLP, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linked irnmunosorbent assay (ELISA), radioim munoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based inununoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson RLP is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983;

[0097] J. Exp. Med. 158:1211-1216).

[0098] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding RLPinclude oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding RLP, or any portions thereof may be cloned into a vector forthe production of an MRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo,Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp. (Cleveland,Ohio)). Suitable reporter molecules or labels, which may be used,include radionuclides, enzymes, fluorescent, chemiluminescent, orchromogenic agents as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

[0099] Host cells transformed with nucleotide sequences encoding RLP maybe cultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeRLP may be designed to contain signal sequences which direct secretionof RLP through a prokaryotic or eukaryotic cell membrane. Otherrecombinant constructions may be used to join sequences encoding RLP tonucleotide sequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and RLP may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingRLP and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMIAC (immobilized metal ion affinitychromatography) as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3: 263-281) while the enterokinase cleavage site provides a meansfor purifying RILP from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll, D.J. et al. (1993;DNA Cell Biol. 12:441-453).

[0100] In addition to recombinant production, fragments of RLP may beproduced by direct peptide synthesis using solid-phase techniques(Merrifield J. (1963) J. Aim Che im Soc. 85:2149-2154). Proteinsynthesis may be performed using manual techniques or by automation.Automated synthesis may be achieved, for example , using AppliedBiosystems 431A Peptide Synthesizer (Perkin Elmer). Various fragments ofRLP may be chemically synthesized separately and combined using chemicalmethods to produce the full length molecule.

[0101] Therapeutics

[0102] RLP shares chemical and structural homology with a human Ras-likeprotein, TC21 (GI 190877) and a mouse Ras-like protein, Rit (GI1656005). Northern analysis shows that the expression of RLP isassociated with cell proliferation and inflammation am .

[0103] Therefore, in one embodiment, RLP or a fragment or derivativethereof may be added to cells to stimulate cell proliferation. Inparticular, RLP may be added to a cell or cells in vivo using deliverymechanisms such as liposomes, viral based vectors, or electroinjectionfor the purpose of promoting regeneration or differentiation of the cellor cells. In addition, RLP may be added to a cell, cell line, tissue ororgan culture in vitro or ex vivo to stimulate cell proliferation foruse in heterologous or autologous transplantation. In some cases, thecell will have been selected for its ability to fight an infection or acancer or to correct a genetic defect in a disease such as sickle cellanemia, D thalassemia, cystic fibrosis, or Huntington's choria.

[0104] In another embodiment, an agonist which is specific for RLP maybe administered to stimulate cell proliferation, as detailed above.

[0105] In still another embodiment, a vector capable of expressing RLP,or a fragment or a derivative thereof, may be administered to stimulatecell proliferation, as detailed above.

[0106] In another embodiment, an antagonist or an inhibitor of RLP, or afragment or a derivative thereof, may be ad ministered to a subject toprevent or treat a disorder associated with cell proliferation.Disorders of cell proliferation include various types of cancerincluding, but not limited to, adenocarcinoma, sarcoma, lymphoma,leukemia, melanoma, myeloma, teratocarcinoma, and in particular, cancersof the bladder, bone, brain, breast, gastrointestinal tract, heart,kidney, liver, lung, ovary, pancreas, paraganglia, parathyroid,prostate, skin, testis, thyroid, and uterus. In one aspect, an antibodyspecific for RLP may be used directly as an antagonist, or indirectly asa targeting or delivery mechanism for bringing a pharmaceutical agent tocells or tissue which express RLP.

[0107] In still another embodiment, a vector expressing thecomplementary sequence or antisense of the polynucleotide encoding RLP,or a fragment or a derivative thereof, may be administered to a subjectto prevent or treat a disorder associated with cell proliferationincluding, but not limited to, those listed above.

[0108] In a further embodiment, an antagonist or an inhibitor of RLP ora fragment or a derivative thereof, may be administered to a subject toprevent or treat inflammation of any type and, in particular, that whichresults from a particular disorder. Such disorders of infla m mationinclude, but are not limited to, AIDS, Addison's disease, allergies,asthma, bronchitis, Crohn's disease, dermatomyositis, diabetes mellitus,emphysema, Graves' disease, irritable bowel syndrome, lupuserythematosus, myasthenia gravis, multiple sclerosis, urethritis,rheumatoid and osteoarthritis, thyroiditis, and ulcerative colitis. Inone aspect, an antibody specific for RLP may be used directly as anantagonist, or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express RLP.

[0109] In another further embodiment, a vector expressing thecomplementary sequence or antisense of the polynucleotide encoding RLP,or a fragment or a derivative thereof, may be administered to a subjectto prevent or treat inflammation of any type including, but not limitedto, those listed above.

[0110] In other embodiments, any of the therapeutic proteins,antagonists, antibodies, agonists, complementary or antisense sequencesor vectors described above may be administered in combination with otherappropriate therapeutic agents. Selection of the appropriate agents foruse in combination therapy may be made by one of ordinary skill in theart, according to conventional pharmaceutical principles. Thecombination of therapeutic agents may act synergistically to effect thetreatment or prevention of the various disorders described above. Usingthis approach, one may be able to achieve therapeutic efficacy withlower dosages of each agent, thus reducing the potential for adverseside effects.

[0111] Antagonists or inhibitors of RLP may be produced using methodswhich are generally known in the art. In particular, purified RLP may beused to produce antibodies or to screen libraries of pharmaceuticalagents to identify those which specifically bind RLP.

[0112] Antibodies to RLP may be generated using methods that are wellknown in the art. Such antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric, single chain, Fab fragments, andfragments produced by a Fab expression library. Neutralizing antibodies,(i.e., those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0113] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith RLP or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

[0114] It is preferred that the peptides, fragments, or oligopeptidesused to induce antibodies to RLP have an amino acid sequence consistingof at least five amino acids, and more preferably at least 10 aminoacids. It is also preferable that they are identical to a portion of theamino acid sequence of the natural protein, and they may contain theentire amino acid sequence of a small, naturally occurring molecule.Short stretches of RLP amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

[0115] Monoclonal antibodies to RLP may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120).

[0116] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceRLP-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobin libraries (BurtonD. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

[0117] Antibodies specific for RLP may also be produced by inducing invivo production in the lymphocyte population or by screening recombinanti m nunoglobulin libraries or panels of highly specific r a y bindingreagents as disclosed in the literature (Orlandi, R. et al. (1989) Proc.Natl. Acad. Sci. 86:

[0118]3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

[0119] Antibody fragments which contain specific binding sites for RLPmay also be generated. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W.D. et al. (1989) Science 254:1275-1281).

[0120] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between RLP and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering RLP epitopes is preferred, but a competitive bindingassay may also be employed (Maddox, supra).

[0121] In another embodiment of the invention, the polynucleotidesencoding RLP, or any fragment thereof, or antisense molecules, may beused for therapeutic purposes. In one aspect, antisense to thepolynucleotide encoding RLP may be used in situations in which it wouldbe desirable to block the transcription of the mRNA. In particular,cells may be transformed with sequences complementary to polynucleotidesencoding RLP. Thus, antisense molecules may be used to modulate RLPactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligomers or largerfragments, can be designed from various locations along the coding orcontrol regions of sequences encoding RLP.

[0122] Expression vectors derived from retro viruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensemolecules complementary to the polynucleotides of the gene encoding RLP.These techniques are described both in Sambrook et al. (supra) and inAusubel et al. (supra).

[0123] Genes encoding RLP can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide or fragment thereof which encodes RLP. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the DNA, suchvectors may continue to transcribe RNA molecules until they are disabledby endogenous nucleases. Transient expression may last for a month ormore with a non-replicating vector and even longer if appropriatereplication elements are part of the vector system

[0124] As mentioned above, modifications of gene expression can beobtained by designing antisense molecules, DNA, RNA, or PNA, to thecontrol regions of the gene encoding RLP, i.e., the promoters,enhancers, and introns. Oligonucleotides derived from the transcriptioninitiation site, e.g., between positions −10 and +10 from the startsite, are preferred. Similarly, inhibition can be achieved using “triplehelix” base-pairing methodology. Triple helix pairing is use ful becauseit causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (Gee, J.E. et al. (1994) In: Huber, B.E. and B. I. Carr, Molecular and Immunologic Approaches, FuturaPublishing Co., Mt. Kisco, N.Y.). The antisense molecules may also bedesigned to block translation of mRNA by preventing the transcript frombinding to ribosomes.

[0125] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding RLP.

[0126] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0127] Antisense molecules and ribozymes of the invention may beprepared by any method known in the art for the synthesis of nucleicacid molecules. These include techniques for chemically synthesizing ooligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences encoding RLP. Such DNA sequences may beincorporated into a wide variety of vectors with suitable RNA polymerasepromoters such as T7 or SP6. Alternatively, these cDNA constructs thatsynthesize antisense RNA constitutively or inducibly can be introducedinto cell lines, cells, or tissues.

[0128] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, andsimilarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0129] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection and by liposomeinjections may be achieved using methods which are well known in theart.

[0130] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0131] An additional embodiment of the invention relates to theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of RLP,antibodies to RLP, mimetics, agonists, antagonists, or inhibitors ofRLP. The compositions may be administered alone or in combination withat least one other agent, such as stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

[0132] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0133] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0134] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0135] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0136] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0137] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0138] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0139] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0140] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes. Thepharmaceutical composition may be provided as a salt and can be formedwith many acids, including but not limited to, hydrochloric, sulfuric,acetic, lactic, tartaric, malic, succi uic, etc. Salts tend to be moresoluble in aqueous or other protonic solvents than are the correspondingfree base forms. In other cases, the preferred preparation may be alyophilized powder which may contain any or all of the following: 1-50mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at a pH range of 4.5to 5.5, that is combined with buffer prior to use.

[0141] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of RLP, such labeling wouldinclude amount, frequency, and method of administration.

[0142] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0143] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0144] A therapeutically effective dose refers to that amount of activeingredient, for example RLP or fragments thereof, antibodies of RLP,agonists, antagonists or inhibitors of RLP, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, ED50/LD50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0145] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0146] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0147] Diagnostics

[0148] In another embodiment, antibodies which specifically bind RLP maybe used for the diagnosis of conditions or diseases characterized byexpression of RLP, or in assays to monitor patients being treated withRLP, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for RLP includemethods which utilize the antibody and a label to detect RLP in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

[0149] A variety of protocols including ELISA, RIA, and FACS formeasuring RLP are known in the art and provide a basis for diagnosingaltered or abnormal levels of RLP expression. Normal or standard valuesfor RLP expression are established by combining body fluids or cellextracts taken from normal mam malian subjects, preferably human, withantibody to RLP under conditions suitable for complex formation. Theamount of standard complex formation may be quantified by variousmethods, but preferably by photometric means. Quantities of RLPexpressed in subject, control, and disease, samples from biopsiedtissues are compared with the standard values. Deviation betweenstandard and subject values establishes the parameters for diagnosingdisease.

[0150] In another embodiment of the invention, the polynucleotidesencoding RLP may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, antisense RNA andDNA molecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofRLP may be correlated with disease. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of RLP, andto monitor regulation of RLP levels during therapeutic intervention.

[0151] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including geno n uc sequences,encoding RLP or closely related molecules, may be used to identifynucleic acid sequences which encode RLP. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intennediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding RLP, alleles, or related sequences.

[0152] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the RLP encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring RLP.

[0153] Means for producing specific hybridization probes for DNAsencoding RLP include the cloning of nucleic acid sequences encoding RLPor RLP derivatives into vectors for the production of nRNA probes. Suchvectors are known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin jbiotin couplingsystems, and the like.

[0154] Polynucleotide sequences encoding RLP may be used for thediagnosis of disorders associated with the expression of RLP. Examplesof such disorders include: various types of cancer such asadenocarcinoma, sarcoma, lymphoma, leukemia, melanoma, myeloma, andcancers of the bladder, bone, brain, breast, gastrointestinal tract,heart, kidney, liver, lung, ovary, pancreas, paraganglia, parathyroid,prostate, skin, testis, thyroid, and uterus; disorders associated withinfla nmation such as AIDS, Addison's disease, allergies, asthma,bronchitis, Crohn's disease, dermatomyositis, diabetes mellitus,emphysema, Graves' disease, irritable bowel syndrome, lupuserythematosus, myasthenia gravis, multiple sclerosis, urethritis,rheumatoid and osteoarthritis, thyroiditis, and ulcerative colitis. Thepolynucleotide sequences encoding RLP may be used in Southern ornorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; or in dip stick, pin, ELISA or chip assays utilizingfluids or tissues from patient biopsies to detect altered RLPexpression. Such qualitative or quantitative methods are well known inthe art.

[0155] In a particular aspect, the nucleotide sequences encoding RLP maybe useful in assays that detect activation or induction of variouscancers, particularly those mentioned above. The nucleotide sequencesencoding RLP may be labeled by standard methods, and added to a fluid ortissue sample from a patient under conditions suitable for the formationof hybridization complexes. After a suitable incubation period, thesample is washed and the signal is quantitated and compared with astandard value. If the amount of signal in the biopsied or extractedsample is significantly altered from that of a comparable controlsample, the nucleotide sequences have hybridized with nucleotidesequences in the sample, and the presence of altered levels ofnucleotide sequences encoding RLP in the sample indicates the presenceof the associated disease. Such assays may also be used to evaluate theefficacy of a particular therapeutic treatment regimen in animalstudies, in clinical trials, or in monitoring the treatment of anindividual patient.

[0156] In order to provide a basis for the diagnosis of diseaseassociated with expression of RLP, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes RLP, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

[0157] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0158] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0159] Additional diagnostic uses for oligonucleotides designed from thesequences encoding RLP may involve the use of PCR. Such oligomers may bechemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation (5′->3′) and another withantisense (3<-5′), employed under optimized conditions foridentification of a specific gene or condition. The same two oligomers,nested sets of oligomers, or even a degenerate pool of oligomers may beemployed under less stringent conditions for detection and/orquantitation of closely related DNA or RNA sequences.

[0160] Methods which may also be used to quantitate the expression ofRLP include radiolabeling or biotinylating nucleotides, coamplificationof a control nucleic acid, and standard curves onto which theexperimental results are interpolated (Melby, P. C. et al. (1993) J.Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor colorimetric response gives rapid quantitation.

[0161] In another embodiment of the invention, the nucleic acidsequences which encode RLP may also be used to generate hybridizationprobes which are useful for mapping the naturally occurring genonicsequence. The sequences may be mapped to a particular chromosome or to aspecific region of the chromosome using well known techniques. Suchtechniques include FISH, FACS, or artificial chromosome constructions,such as yeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

[0162] FISH (as described in Verma et al. (1988) Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York, N.Y.) may becorrelated with other physical chromosome mapping techniques and geneticmap data. Examples of genetic map data can be found in the 1994 GenomeIssue of Science (265:1981fill). Correlation between the location of thegene encoding RLP on a physical chromosomal map and a specific disease,or predisposition to a specific disease, may help delimit the region ofDNA associated with that genetic disease. The nucleotide sequences ofthe subject invention may be used to detect differences in genesequences between normal, carrier, or affected individuals.

[0163] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the nuriber or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (Gatti, R. A.et al. (1988) Nature 336:577-580), any sequences mapping to that areamay represent associated or regulatory genes for further investigation.The nucleotide sequence of the subject invention may also be used todetect differences in the chromosomal location due to translocation,inversion, etc. among normal, carrier, or affected individuals.

[0164] In another embodiment of the invention, RLP, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, between RLPand the agent being tested, may be measured.

[0165] Another technique for drug screening which may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication WO84/03564. In this method, as applied to RLP large numbersof different small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with RLP, or fragments thereof, and washed. Bound RLP is thendetected by methods well known in the art. Purified RLP can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

[0166] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding RLPspecifically compete with a test compound for binding RLP. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with RLP.

[0167] In additional enibodiments, the nucleotide sequences which encodeRLP may be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

[0168] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0169] I UTRSNOT02 cDNA Library Construction

[0170] The normal tissue used for uterus cDNA library construction wasobtained from a 34 year-old female. The frozen tissue was homogenizedand lysed using a Brinkmann Homogenizer Polytron PT-3000 (Brinkma nmInstruments, Westbury, N.J.). The reagents and extraction procedureswere used as supplied in the Stratagene RNA Isolation Kit (Catalog#200345; Stratagene). The lysate was centrifuged over a 5.7 M CsClcushion using a Beckman SW28 rotor in a Beckman L8-70M Ultracentrifuge(Beckman Instruments) for 18 hours at 25,000 rpm at ambient temperature.The RNA was extracted three times with acid phenol pH 4.0, precipitatedusing 0.3 M sodium acetate and 2.5 volumes of ethanol, resuspended inwater and DNase treated for 15 min at 37° C. The RNA was isolated usingthe QIAGEN OLIGOTEX kit (QIAGEN Inc, Chatsworth, Calif.) and used toconstruct the cDNA library.

[0171] The RNA was handled according to the recommended protocols in theSuperScript Plasmid System for cDNA Synthesis and Plasmid Clo uing(Catalog #18248-013, Gibco/BRL, Gaithersburg, MD), cDNAs werefractionated on a Sepharose CL4B column (Catalog #275105, Pharmacia) andligated into the PSPORT 1 plasmid. The plasmid was subsequentlytransformed into DH5α competent cells (Catalog #18258-012, Gibco/BRL).

[0172] II Isolation and Sequencing of cDNA Clones

[0173] Plasmid DNA was released from the cells and purified using theMiniprep Kit (Catalog #77468; Advanced Genetic Technologies Corporation,Gaithersburg, MD). This kit consists of a 96-well block with reagentsfor 960 purifications. The recommended protocol was employed except forthe following changes: 1) the 96 wells were each filled with only 1 n mof sterile Terrific Broth (Catalog #22711, Gibco/BRL) with carbenicillinat 25 mg/L and glycerol at 0.4%; 2) the bacteria were cultured for 24hours after the wells were inoculated and then lysed with 60 μl of lysisbuffer; 3) a centrifugation step employing the Beckman GS-6R rotor at2900 rpm for 5 minutes was performed before the contents of the blockwere added to the primary filter plate; and 4) the optional step ofadding isopropanol to TRIS buffer was not routinely performed. After thelast step in the protocol, samples were transferred to a Beckman 96-wellblock for storage.

[0174] The cDNAs were sequenced by the method of Sanger F and AR Coulson(1975; J Mol Biol 94:441fill), using a Hamiton Micro Lab 2200 (Hamilton,Reno, Nev.) in combination with four Peltier Thermal Cyclers (PTC200from MJ Research, Watertown, Mass.) and Applied Biosystems 377 or 373DNA Sequencing Systems, and the reading frame was determined.

[0175] III Homology Searching of cDNA Clones and Their Deduced Proteins

[0176] After the reading frame was determined, the nucleotide sequencesof the Sequence Listing or amino acid sequences deduced from them wereused as query sequences against databases such as GenBank, SwissProt,BLOCKS, and Pima II. These databases which contain previously identifiedand annotated sequences, were searched for regions of homology(similarity) using BLAST, which stands for Basic Local Alignment SearchTool (Altschul (1993) supra, Altschul (1990) supra).

[0177] BLAST produced alignments of both nucleotide and amino acidsequences to determine sequence si milarity. Because of the local natureof the alignments, BLAST was especially useful in determining exactmatches or in identifying homologs which may be of prokaryotic(bacterial) or eukaryotic (animal, fungal, or plant) origin. Otheralgorithms such as the one described in Smith et al. (1992, ProteinEngineering 5:35-51), incorporated herein by reference, could have beenused when dealing with pri try sequence patterns and secondary structuregap penalties. The sequences disclosed in this application have lengthsof at least 49 nucleotides, and no more than 12% uncalled bases (where Nis recorded rather than A, C, G, or T).

[0178] The BLAST approach, as detailed in Karlin et al. (supra) andincorporated herein by reference, searched for matches between a querysequence and a database sequence. BLAST evaluated the statisticalsignificance of any matches found, and reported only those matches thatsatisfy the user-selected threshold of significance. In thisapplication, threshold was set at 10-25 for nucleotides and 10⁻¹⁴ forpeptides.

[0179] IV Northern Analysis

[0180] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound (Sambrook et al., supra).

[0181] Analogous computer techniques using BLAST (Altschul, S. F. 1993and 1990, supra) are used to search for identical or related moleculesin nucleotide databases such as GenBank or the LIFESEQ™ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

[0182] The basis of the search is the product score which is defined as:$\frac{\% \quad {sequence}\quad {identity} \times \quad \% \quad {maximum}\quad {BLAST}\quad {score}}{100}$

[0183] The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. Forexample, with a product score of 40, the match will be exact within a1-2% error; and at 70, the match will be exact. Homologous molecules areusually identified by selecting those which show product scores between15 and 40, although lower scores may identify related molecules.

[0184] The results of northern analysis are reported as a list oflibraries in which the transcript encoding RLP occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0185] V Extension of RLP-Encoding Polynucleotides

[0186] Nucleic acid sequence of Incyte clone 683101 or SEQ ID NO:2 isused to design oligonucleotide primers for extending a partialnucleotide sequence to full length or for obtaining 5′ or 3′, intron orother control sequences from genomic libraries. One primer issynthesized to initiate extension in the antisense direction (XLR) andthe other is synthesized to extend sequence in the sense direction(XLF). Primers are used to facilitate the extension of the knownsequence “outward,” generating amplicons containing new, unknownnucleotide sequence for the region of interest. The initial primers aredesigned from the cDNA using OLIGO 4.06 (National Biosciences), oranother appropriate program, to be 22-30 nucleotides in length, to havea GC content of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. Any stretch of nucleotides which wouldresult in hairpin structures and primer-primer di rerizations isavoided.

[0187] The original, selected cDNA libraries, or a human genomic libraryare used to extend the sequence; the latter is most useful to obtain 5′upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0188] By following the instructions for the XL-PCR kit (Perkin Ehner)and thoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier Thermal Cycler (PTC200; M. J. Research,Watertown, Mass.) and the following parameters: Step 1 94° C. for 1 min(initial denaturation) Step 2 65° C. for 1 min Step 3 68° C. for 6 minStep 4 94° C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C. for 7 minStep 7 Repeat step 4-6 for 15 additional cycles Step 8 94° C. for 15 secStep 9 65° C. for 1 min Step 10 68° C. for 7:15 min Step 11 Repeat step8-10 for 12 cycles Step 12 72° C. for 8 min Step 13 4° C. (and holding)

[0189] A 5-10 μl aliquot of the reaction mixture is analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine whichreactions were successful in extending the sequence.Bands thought to contain the largest products are selected and removedfrom the gel. Further purification involves using a commercial gelextraction method such as QIAQUICK (QIAGEN Inc., Chatsworth, Calif.).After recovery of the DNA, Klenow enzyme is used to trimsingle-stranded, nucleotide overhangs creating blunt ends whichfacilitate religation and clo rn ig.

[0190] After ethanol precipitation, the products are redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 al of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook et al., supra)containing 2× Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2× Carbmedium placed in an individual well of an appropriate,commercially-available, sterile 96-well microtiter plate. The followingday, 5 μl of each overnight culture is transferred into a non-sterile96-well plate and after dilution 1:10 with water, 5 μl of each sample istransferred into a PCR array.

[0191] For PCR amplification, 18 Al of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionare added to each well. Amplification is performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7 4° C. (andholding)

[0192] Aliquots of the PCR reactions are run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products arecompared to the original partial cDNAs, and appropriate clones areselected, ligated into plasmid, and sequenced.

[0193] VI Labeling and Use of Hybridization Probes

[0194] Hybridization probes derived from SEQ ID NO:2 are employed toscreen cDNAs, geno rnic DNAs, or m RNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 μCi of [γ-³²P] adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DuPont NEN ®, Boston, MA). Thelabeled oligonucleotides are substantially purified with Sephadex G-25superfine resin column (Pharmacia & Upjohn). A portion containing 10⁷counts per minute of each of the sense and antisense oligonucleotides isused in a typical membrane based hybridization analysis of human genornic DNA digested with one of the following endonucleases (Ase I, Bgl II,Eco RI, Pst I, Xba 1, or Pvu II; DuPont NEN®).

[0195] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (Nytran Plus, Schleicher &Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1 × salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoh nagercassette (Molecular Dyna mics, Sunnyvale, Calif.) for several hours,hybridization patterns are compared visually.

[0196] VII Antisense or Complementary Sequences

[0197] Antisense molecules or nucleic acid sequences complementary tothe RLP-encoding sequence, or any part thereof, are used to inhibit invivo or in vitro expression of naturally occurring RLP. Although use ofantisense oligonucleotides, comprising about 20 base-pairs, isspecifically described, essentially the same procedure is used withlarger cDNA fragments. An oligonucleotide based on the coding sequencesof RLP, as shown in FIGS. 1A, 1B and 1C, is used to inhibit expressionof naturally occurring RLP. The complementary oligonucleotide isdesigned from the most unique 5′ sequence as shown in FIGS. 1A, 1B and1C and used either to inhibit transcription by preventing promoterbinding to the upstream nontranslated sequence or translation of anRLP-encoding transcript by preventing the ribosome from binding. Usingan appropriate portion of the signal and 5′ sequence of SEQ ID NO:2, aneffective antisense oligonucleotide includes any 15-20 nucleotidesspanning the region which translates into the signal or 5′ codingsequence of the polypeptide as shown in FIGS. 1A, 1B and 1C.

[0198] VIII Expression of RLP

[0199] Expression of RLP is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the PSPORT 1 cloning vector, previously used for thegeneration of the cDNA library, is used to express RLP in E. coli.Upstream of the cloning site, this vector contains a promoter forB-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

[0200] Induction of an isolated, transformed bacterial strain with IPTGusing standard methods produces a fusion protein which consists of thefirst eight residues of β-galactosidase, about 5 to 15 residues oflinker, and the full length protein. The signal residues direct thesecretion of RLP into the bacterial growth media which can be useddirectly in the following assay for activity.

[0201] IX Demonstration of RLP Activity

[0202] RLP can be expressed in a mammalian cell line such as 293T bytransfecting with an eukaryotic expression vector encoding RLP.Eukaryotic expression vectors are commercially available, and thetechniques to introduce them into cells are well known to those skilledin the art. A small amount of a second plasmid, wh ich expresses any oneof a number of reporter genes such as γ-galactosidase, is co-transformedinto the cells in order to allow rapid identification of those cellswhich have taken up and expressed the foreign DNA. The cells arecultured in a defined synthetic medium with concentrations of GTP for atleast 48 hours after transformation to allow expression and accumulationof RLP and γ-galactosidase.

[0203] Transformed cells expressing γ-galactosidase are stained bluewhen a suitable calorimetric substrate is added to the culture mediaunder conditions that are well known in the art. Increasingconcentrations of GTP induces increasing numbers of reporter genepositive cells (Ren, M. et al. (1996) Proc. Natl. Acad. Sci. 93:5151-5155). GTP-treated cells which were not transformed with the RLPexpression vector are used as controls as are RLP transfected cellscultured without supplemental GTP.

[0204] X Production of RLP Specific Antibodies

[0205] RLP that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols.

[0206] The amino acid sequence deduced from SEQ ID NO:2 is analyzedusing DNASTAR software (DNASTAR Inc) to determine regions of highimmunogenicity and a corresponding oligopolypeptide is synthesized andused to raise antibodies by means known to those of skill in the art.Selection of appropriate epitopes, such as those near the C-terminus orin hydrophilic regions, is described by Ausubel et al. (supra), andothers.

[0207] Typically, the oligopeptides are 15 residues in length,synthesized using an Applied Biosystems Peptide Synthesizer Model 431Ausing fmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH,Sigma, St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel et al.,supra). Rabbits are immunized with the o ligopeptide-KlI complex incomplete Freund's adjuvant. The resulting antisera are tested forantipeptide activity, for example, by binding the peptide to plastic,blocking with 1% BSA, reacting with rabbit antisera, washing, andreacting with radioiodinated, goat anti-rabbit IgG.

[0208] XI Puirification of Naturally Occurring RLP Using SpecificAntibodies

[0209] Naturally occurring or recombinant RLP is substantially purifiedby immunoaffinity chromatography using antibodies specific for RLP. Animmunoaffinity column is constructed by covalently coupling RLP antibodyto an activated chromatographic resin, such as CnBr-activated Sepharose(Pharmacia & Upjohn). After the coupling, the resin is blocked andwashed according to the manufacturer's instructions.

[0210] Media containing RLP is passed over the i munoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of RLP (e.g., high ionic strength buffers in the presence ofdetergent). The colun n is eluted under conditions that disruptantibodylRLP binding (eg, a buffer of pH 2-3 or a high concentration ofa chaotrope, such as urea or thiocyanate ion), and RLP is collected.

[0211] XII Identification of Molecules Which Interact with RLP

[0212] RLP or biologically active fragments thereof are labeled with¹²⁵I Bolton-Hunter reagent (Bolton et al. (1973) Biocheim J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled RLP, washed and any wells withlabeled RLP complex are assayed. Data obtained using differentconcentrations of RLP are used to calculate values for the number,affinity, and association of RLP with the candidate molecules.

[0213] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 3 214 amino acids amino acid single linear COLNTUT16 2791521 1 Met AsnCys Lys Glu Gly Thr Asp Ser Ser Cys Gly Cys Arg Gly Asn 1 5 10 15 AspGlu Lys Lys Met Leu Lys Cys Val Val Val Gly Asp Gly Ala Val 20 25 30 GlyLys Thr Cys Leu Leu Met Ser Tyr Ala Asn Asp Ala Phe Pro Glu 35 40 45 GluTyr Val Pro Thr Val Phe Asp His Tyr Ala Val Thr Val Thr Val 50 55 60 GlyGly Lys Gln His Leu Leu Gly Leu Tyr Asp Thr Ala Gly Gln Glu 65 70 75 80Asp Tyr Asn Gln Leu Arg Pro Leu Ser Tyr Pro Asn Thr Asp Val Phe 85 90 95Leu Ile Cys Phe Ser Val Val Asn Pro Ala Ser Tyr His Asn Val Gln 100 105110 Glu Glu Trp Val Pro Glu Leu Lys Asp Cys Met Pro His Val Pro Tyr 115120 125 Val Leu Ile Gly Thr Gln Ile Asp Leu Arg Asp Asp Pro Lys Thr Leu130 135 140 Ala Arg Leu Leu Tyr Met Lys Glu Lys Pro Leu Thr Tyr Glu HisGly 145 150 155 160 Val Lys Leu Ala Lys Ala Ile Gly Ala Gln Cys Tyr LeuGlu Cys Ser 165 170 175 Ala Leu Thr Gln Lys Gly Leu Lys Ala Val Phe AspGlu Ala Ile Leu 180 185 190 Thr Ile Phe His Pro Lys Lys Lys Lys Lys ArgCys Ser Glu Gly His 195 200 205 Ser Cys Cys Ser Ile Ile 210 2964 basepairs nucleic acid single linear COLNTUT16 2791521 2 CCATGTTAGATGTGACTTGG AAAATGAGAA AGATTTAGCA AAATTCCACC GTGTCTTTTG 60 CCAGGCTAGAGACAGGGAGA GCAGAGTAAA ACCCTCAGGC TGCTGAAATT TCTAGGCTGT 120 TAGGAAGCCCCTCGAATTCT GTGAAAATGA GGGTTTCTTA ACTCACACTG AGAGCGGAAA 180 GGGGCAGACCCTTTTCATAA CTCCCTCAAG TGTGTGTTAC CTTTCTTTAC CAGCATGGTA 240 AGCAACAGGACATATCCCAG CCTCGGACAT GTCTGTATGA TCCAAGGTAC CCAAAGTCAG 300 ACAGAGTAAACTCAAGCCTG GCACTGGCTT TCTGCCGCTT CATGTGCTTT GGAAAAAGCA 360 GGAGAAGCAATAGCAGCAGG AGTCCCCAGC AGCTGGAGCC GCAAGAATGA ACTGCAAAGA 420 GGGAACTGACAGCAGCTGCG GCTGCAGGGG CAACGACGAG AAGAAGATGT TGAAGTGTGT 480 GGTGGTGGGGGACGGTGCCG TGGGGAAAAC CTGCCTGCTG ATGAGCTACG CCAACGACGC 540 CTTCCCAGAGGAATACGTGC CCACTGTGTT TGACCACTAT GCAGTTACTG TGACTGTGGG 600 AGGCAAGCAACACTTGCTCG GACTGTATGA CACCGCGGGA CAGGAGGACT ACAACCAGCT 660 GAGGCCACTCTCCTACCCCA ACACGGATGT GTTTTTGATC TGCTTCTCTG TCGTAAACCC 720 TGCCTCTTACCACAATGTCC AGGAGGAATG GGTCCCCGAG CTCAAGGACT GCATGCCTCA 780 CGTGCCTTATGTCCTCATAG GGACCCAGAT TGATCTCCGT GATGACCCAA AAACCTTGGC 840 CCGTTTGCTGTATATGAAAG AGAAACCTCT CACTTACGAG CATGGTGTGA AGCTCGCAAA 900 AGCGATCGGAGCACAGTGCT ACTTGGAATG TTCAGCTCTG ACTCAGAAAG GTCTCAAAGC 960 GGTTTTTGATGAAGCAATCC TCACCATTTT CCACCCCAAG AAAAAGAAGA AACGCTGTTC 1020 TGAGGGTCACAGCTGCTGTT CAATTATCTG AGGTTGTCTG GGACCTGCCT CCACCCCATC 1080 CAGGGATGAGAATGGCAGCC AATCTCTGTG GCCAAGCTCC AGCCAAAAAG GAGGGCACGA 1140 CCAGAAAGGAACTCCCTTTG CACGGAGGCT TGCCCCATCA CCCTCTGAGC CCTCCCAACA 1200 CAGCACACTAGTCAGCCCAC TGCCACGACC TCCCTGCCAG CCAGAAGCAT CCGTACTGCA 1260 CGCTGTCTGAGAATGCTGGG CCTGGATTGC AGACAGTGCC GCTGCTGATC GCATCAAAAA 1320 CAAAGTCAAAGGCCATCTCA CATTTTACAA ATCCCCAGCT CATGAACGTG AAGCTGATAG 1380 GAAATCACCCCAGGGAACCC GAAAAAGAAA CTTGATTCCT CTATTGCTGG CCTTACTTGA 1440 TGTCTTTTATAAAACTTGGG ACTACAATAC TAACCTTTTT TTCTGAATCT GCTGTTCTAC 1500 CCATGTGTCTCACATTCATT TGTATTATTT CAAGAAATGT ACTAATTTCC AGTTCACTCA 1560 GGCCTTACTAATCCATACCA AATTAGCCTA AAGACAAGGC ATTTTATATT CATTTCTATT 1620 TTCAGCATGTTTCTACCAAA GCTATTAGAA CCAACACGTA CCTCTGAATG CCCGATTATA 1680 AGAAGACATGAGAAGACTTT AAAAGTTTTG GAAATTTACA GAGCCATGAT TTTTGAACCT 1740 AATTGAAAGAAAACCATCTG AATTGTTGCA GGTCCACATT TTTGCCAAAG ATACACTCTA 1800 TAGATGCTTAGTAGTGGCCT GATTTTTTTC CATGTATTGC CACGACAAAC TAAAAATGAA 1860 CTGTGTTTAAGAATGTAGTA TTTCTGTTTT TCATCCAAGT TGATTGGGGG AAGAATATGG 1920 CAGGATCCATCTTTTACAGT ATTTTGTATT CAGTAAAGTG GACATTCCTG CTCCTCCCTT 1980 CCCCCATTGCATGCCCTCTT CCTCCCTTGA TTTCACTTTC TCTCATGCCC GGATCCTTTT 2040 ATTCTCCCCAGTTATAACCC AGTTATAAAA GAAAGATCTG AGCATAAAGA TACGTGTTTA 2100 AAAATAACTAAAAGTAAAGG AAAGTGCCTT AATTTTTCTA TTTGCTTCAA CTGAAAGTGC 2160 TTCTCAGCTCGCCCCATGTA AGTTCTCATT CCATGTAAAT GACATTTTCC AGTTACAACT 2220 GGTACTGAGATTTTGCCTCT CTCTTTCCTT ACTCATCCTC CCAAATGTCT TTGTGGGAGC 2280 CATATCAGTGGATACCAAGC TCTGTATCCA TTTGTCCCCT GCCCTCCACA ATGTGTGACA 2340 TAGAACAGGGACTTTGGCCC TGGGAAAGCA AAAGCTCCCA GTAAGGAATC CTGTGCCCAA 2400 TGATGTAAAACAATTCCAAA CATCCAGGAA TTTTTGTATC ATAGAGCGAA TTACTTCCTA 2460 TCTTTTCATTAGAGGCTATG AGGACTTCTA ATTAGTCTTA GTTGCTTATA AGTGCCCTGG 2520 AATCACCCAGGTAGGCACTT AATTTTTTTT TCAGTTGCAT GAGCAAAGTG CTTCTTAGTA 2580 GTGTGAAATTACAACAACTT TAAGACTTTC CAGATTCAAG CTCCCACTGT TGGAAAAAGC 2640 CAGCCTTTCTAATCTCTTCT GCTACTGGAA TAAGCACTTA AGAATTGCGT GATAGCCAGG 2700 CACCGTGGCTCATGCCTGTA ATCCCAACAC TTAGGGAGGC TGAGGTGGGT GGGCCGCTTG 2760 AGCTCAGGAGTTCAAGACCA GCCTGGGTAA TATAGTGAGA TCCTGTGTCT CTATAAAAAA 2820 ATTAAAAATTAGTCAGTTGT AGTGACACAT ACCTGTAGTC CCAGCTACTC AGGAGGCTGA 2880 GGTGGAAGGATCACTTGAGC CCAGAAGGTA AGGCTGCAGT GAGCTGTGAC TGTGCCACTA 2940 CACTCCAGCCTGAGTGACAG AGAA 2964 213 amino acids amino acid single linear GenBank190881 3 Met Pro Gly Ala Gly Arg Ser Ser Met Ala His Gly Pro Gly Ala Leu1 5 10 15 Met Leu Lys Cys Val Val Val Gly Asp Gly Ala Val Gly Lys ThrCys 20 25 30 Leu Leu Met Ser Tyr Ala Asn Asp Ala Phe Pro Glu Glu Tyr ValPro 35 40 45 Thr Val Phe Asp His Tyr Ala Val Ser Val Thr Val Gly Gly LysGln 50 55 60 Tyr Leu Leu Gly Leu Tyr Asp Thr Ala Gly Gln Glu Asp Tyr AspArg 65 70 75 80 Leu Arg Pro Leu Ser Tyr Pro Met Thr Asp Val Phe Leu IleCys Phe 85 90 95 Ser Val Val Asn Pro Ala Ser Phe Gln Asn Val Lys Glu GluTrp Val 100 105 110 Pro Glu Leu Lys Glu Tyr Ala Pro Asn Val Pro Phe LeuLeu Ile Gly 115 120 125 Thr Gln Ile Asp Leu Arg Asp Asp Pro Lys Thr LeuAla Arg Leu Asn 130 135 140 Asp Met Lys Glu Lys Pro Ile Cys Val Glu GlnGly Gln Lys Leu Ala 145 150 155 160 Lys Glu Ile Gly Ala Cys Cys Tyr ValGlu Cys Ser Ala Leu Thr Gln 165 170 175 Lys Gly Leu Lys Thr Val Phe AspGlu Ala Ile Ile Ala Ile Leu Thr 180 185 190 Pro Lys Lys His Thr Val LysLys Arg Ile Gly Ser Arg Cys Ile Asn 195 200 205 Cys Cys Leu Ile Thr 210

What is claimed is:
 1. An isolated polypeptide selected from the groupconsisting of: a) a polypeptide comprising an amino acid sequence of SEQID NO:1, b) a polypeptide comprising a naturally occurring amino acidsequence at least 90% identical to an amino acid sequence of SEQ IDNO:1, c) a biologically active fragment of a polypeptide having an aminoacid sequence of SEQ ID NO:l, and d) an immunogenic fragment of apolypeptide having an amino acid sequence of SEQ ID NO:1.
 2. An isolatedpolypeptide of claim 1 comprising an amino acid sequence of SEQ ID NO:1.3. An isolated polynucleotide encoding a polypeptide of claim
 1. 4. Anisolated polynucleotide encoding a polypeptide of claim
 2. 5. Anisolated polynucleotide of claim 4 comprising a polynucleotide sequenceof SEQ ID NO:2.
 6. A recombinant polynucleotide comprising a promotersequence operably linked to a polynucleotide of claim
 3. 7. A celltransformed with a recombinant polynucleotide of claim
 6. 8. Atransgenic organism comprising a recombinant polynucleotide of claim 6.9. A method of producing a polypeptide of claim 1, the methodcomprising: a) culturing a cell under conditions suitable for expressionof the polypeptide, wherein said cell is transformed with a recombinantpolynucleotide, and said recombinant polynucleotide comprises a promotersequence operably linked to a polynucleotide encoding the polypeptide ofclaim 1, and b) recovering the polypeptide so expressed.
 10. A method ofclaim 9, wherein the polypeptide comprises an amino acid sequence of SEQID NO:1.
 11. An isolated antibody which specifically binds to apolypeptide of claim
 1. 12. An isolated polynucleotide selected from thegroup consisting of: a) a polynucleotide comprising a polynucleotidesequence of SEQ ID NO:2, b) a polynucleotide comprising a naturallyoccurring polynucleotide sequence at least 90% identical to apolynucleotide sequence selected from the group consisting of SEQ IDNO:2, c) a polynucleotide complementary to a polynucleotide of a), d) apolynucleotide complementary to a polynucleotide of b), and e) an RNAequivalent of a)-d).
 13. An isolated polynucleotide comprising at least60 contiguous nucleotides of a polynucleotide of claim
 12. 14. A methodof detecting a target polynucleotide in a sample, said targetpolynucleotide having a sequence of a polynucleotide of claim 12, themethod comprising: a) hybridizing the sample with a probe comprising atleast 20 contiguous nucleotides comprising a sequence complementary tosaid target polynucleotide in the sample, and which probe specificallyhybridizes to said target polynucleotide, under conditions whereby ahybridization complex is formed between said probe and said targetpolynucleotide or fragments thereof, and b) detecting the presence orabsence of said hybridization complex, and, optionally, if present, theamount thereof.
 15. A method of claim 14, wherein the probe comprises atleast 60 contiguous nucleotides.
 16. A method of detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 12, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 17. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 18. Acomposition of claim 17, wherein the polypeptide comprises an amino acidsequence of SEQ ID NO:
 1. 19. A method for treating a disease orcondition associated with decreased expression of functional RLP,comprising administering to a patient in need of such treatment thecomposition of claim
 17. 20. A method of screening a compound foreffectiveness as an agonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting agonist activity in the sample.
 21. Acomposition comprising an agonist compound identified by a method ofclaim 20 and a pharmaceutically acceptable excipient.
 22. A method fortreating a disease or condition associated with decreased expression offunctional RLP, comprising administering to a patient in need of suchtreatment a composition of claim
 21. 23. A method of screening acompound for effectiveness as an antagonist of a polypeptide of claim 1,the method comprising: a) exposing a sample comprising a polypeptide ofclaim 1 to a compound, and b) detecting antagonist activity in thesample.
 24. A composition comprising an antagonist compound identifiedby a method of claim 23 and a pharmaceutically acceptable excipient. 25.A method for treating a disease or condition associated withoverexpression of functional RLP, comprising administering to a patientin need of such treatment a composition of claim
 24. 26. A method ofscreening for a compound that specifically binds to the polypeptide ofclaim 1, the method comprising: a) combi n ig the polypeptide of claim 1with at least one test compound under suitable conditions, and b)detecting binding of the polypeptide of claim 1 to the test compound,thereby identifying a compound that specifically binds to thepolypeptide of claim
 1. 27. A method of screening for a compound thatmodulates the activity of the polypeptide of claim 1, the methodcomprising: a) combining the polypeptide of claim 1 with at least onetest compound under conditions per m issive for the activity of thepolypeptide of claim 1, b) assessing the activity of the polypeptide ofclaim 1 in the presence of the test compound, and c) comparing theactivity of the polypeptide of claim 1 in the presence of the testcompound with the activity of the polypeptide of claim 1 in the absenceof the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 28. A method of screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a sequence of claim 5, the method comprising:a) exposing a sample comprising the target polynucleotide to a compound,under conditions suitable for the expression of the targetpolynucleotide, b) detecting altered expression of the targetpolynucleotide, and c) comparing the expression of the targetpolynucleotide in the presence of varying amounts of the compound and inthe absence of the compound.
 29. A method of assessing toxicity of atest compound, the method comprising: a) treating a biological samplecontaining nucleic acids with the test compound, b) hybridizing thenucleic acids of the treated biological sample with a probe comprisingat least 20 contiguous nucleotides of a polynucleotide of claim 12 underconditions whereby a specific hybridization complex is formed betweensaid probe and a target polynucleotide in the biological sample, saidtarget polynucleotide comprising a polynucleotide sequence of apolynucleotide of claim 12 or fragment thereof, c) quantifying theamount of hybridization complex, and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 30. Adiagnostic test for a condition or disease associated with theexpression of RLP in a biological sample, the method comprising: a)combining the biological sample with an antibody of claim 11, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex, and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 31. The antibody of claim 11, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂fragment, or e) a humanized antibody.
 32. Acomposition comprising an antibody of claim 11 and an acceptableexcipient.
 33. A method of diagnosing a condition or disease associatedwith the expression of RLP in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 32. 34. Acomposition of claim 32, wherein the antibody is labeled.
 35. A methodof diagnosing a condition or disease associated with the expression ofRLP in a subject, comprising administering to said subject an effectiveamount of the composition of claim
 34. 36. A method of preparing apolyclonal antibody with the specificity of the antibody of claim 11,the method comprising: a) immunizing an animal with a polypeptideconsisting of an amino acid sequence of SEQ ID NO: 1, or an immunogenicfragment thereof, under conditions to elicit an antibody response, b)isolating antibodies from said animal, and c) screening the isolatedantibodies with the polypeptide, thereby identif y ig a polyclonalantibody which specifically binds to a polypeptide comprising an aminoacid sequence of SEQ ID NO:
 1. 37. A polyclonal antibody produced by amethod of claim
 36. 38. A composition comprising the polyclonal antibodyof claim 37 and a suitable carrier.
 39. A method of making a monoclonalantibody with the specificity of the antibody of claim 11, the methodcomprising: a) immunizing an animal with a polypeptide consisting of anamino acid sequence of SEQ ID NO: 1, or an immunogenic fragment thereof,under conditions to elicit an antibody response, b) isolating antibodyproducing cells from the animal, c) fusing the antibody producing cellswith immortalized cells to form monoclonal antibody-producing hybridomacells, d) culturing the hybridoma cells, and e) isolating from theculture monoclonal antibody which specifically binds to a polypeptidecomprising an amino acid sequence of SEQ ID NO:
 1. 40. A monoclonalantibody produced by a method of claim
 39. 41. A composition comprisingthe monoclonal antibody of claim 40 and a suitable carrier.
 42. Theantibody of claim 11, wherein the antibody is produced by screening aFab expression library.
 43. The antibody of claim 11, wherein theantibody is produced by screening a recombinant immunoglobulin library.44. A method of detecting a polypeptide comprising an amino acidsequence of SEQ ID NO:1 in a sample, the method comprising: a)incubating the antibody of claim 11 with a sample under conditions toallow specific binding of the antibody and the polypeptide, and b)detecting specific binding, wherein specific binding indicates thepresence of a polypeptide comprising an amino acid sequence of SEQ IDNO:1 in the sample.
 45. A method of purifying a polypeptide comprisingan amino acid sequence of SEQ ID NO:1 from a sample, the methodcomprising: a) incubating the antibody of claim 11 with a sample underconditions to allow specific binding of the antibody and thepolypeptide, and b) separating the antibody from the sample andobtaining the purified polypeptide comprising an amino acid sequence ofSEQ ID NO: I.
 46. A microarray wherein at least one element of themicroarray is a polynucleotide of claim
 13. 47. A method of generatingan expression profile of a sample which contains polynucleotides, themethod comprising: a) labeling the polynucleotides of the sample, b)contacting the elements of the microarray of claim 46 with the labeledpolynucleotides of the sample under conditions suitable for theformation of a hybridization complex, and c) quantifying the expressionof the polynucleotides in the sample.
 48. An array comprising differentnucleotide molecules affixed in distinct physical locations on a solidsubstrate, wherein at least one of said nucleotide molecules comprises afirst oligonucleotide or polynucleotide sequence specificallyhybridizable with at least 30 contiguous nucleotides of a targetpolynucleotide, and wherein said target polynucleotide is apolynucleotide of claim
 12. 49. An array of claim 48, wherein said firstoligonucleotide or polynucleotide sequence is completely complementaryto at least 30 contiguous nucleotides of said target polynucleotide. 50.An array of claim 48, wherein said first oligonucleotide orpolynucleotide sequence is completely complementary to at least 60contiguous nucleotides of said target polynucleotide.
 51. An array ofclaim 48, wherein said first oligonucleotide or polynucleotide sequenceis completely complementary to said target polynucleotide.
 52. An arrayof claim 48, which is a microarray.
 53. An array of claim 48, furthercomprising said target polynucleotide hybridized to a nucleotidemolecule comprising said first oligonucleotide or polynucleotidesequence.
 54. An array of claim 48, wherein a linker joins at least oneof said nucleotide molecules to said solid substrate.
 55. An array ofclaim 48, wherein each distinct physical location on the substratecontains multiple nucleotide molecules, and the multiple nucleotidemolecules at any single distinct physical location have the samesequence, and each distinct physical location on the substrate containsnucleotide molecules having a sequence which differs from the sequenceof nucleotide molecules at another distinct physical location on thesubstrate.
 56. A polypeptide of claim 1, comprising the a tnino acidsequence of SEQ ID NO:
 1. 57. A polynucleotide of claim 12, comprisingthe polynucleotide sequence of SEQ ID NO:2.